Journal of the American Chemical Society
Article
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culture broth. Therefore, to deliver synthetic NAD analogs
should be manageable. Nonspecific interactions between the
synthetic cofactor and other cellular components are among the
other concerns to be addressed. Nonetheless, the accessibility
of such bioorthogonal redox systems should provide unique
tools for synthetic biology to assemble pathway-specific redox
chemistry and for systems biology to elucidate the contribu-
tions of individual redox steps in the metabolic network.
High-Throughput Screening of ME Variants. Recombinant
E. coli colonies from the libraries were grown on LB agar plates
−
1
containing 0.5 mM of IPTG and 50 μg mL of kanamycin. After
incubated at 30 °C for 36 h, the colonies were picked to a 96-well
plate. Cells were resuspended in 100 μL of lysis buffer (50 mM of
−1
HEPES, 1 mg mL of lysozyme, 1% Triton X100, and pH of 7.5) and
lysed by shaking at 37 °C for 1 h. Samples were centrifugation at 4000
g for 15 min, and supernatants were collected and stored at 4 °C.
Assays were performed by transferring 10 μL of each supernatant to 90
μL of reaction buffer (50 mM of HEPES, 3 mM of L-malate, 5 mM of
MnCl , 0.05 mM of NAD or NFCD, 0.1 mM of nitro blue tetrazolium,
2
EXPERIMENTAL PROCEDURES
■
0
.025 mM of phenazine methosulfate, and pH of 7.2). After being
All reagents and starting materials were used as obtained from
commercial suppliers (Sigma, ABCR, ACROS) unless otherwise
indicated. Commercial nucleotides were phosphorylated as previously
reported to give the corresponding monophosphates. NMN was
prepared by hydrolysis of NAD as reported.
incubated at 37 °C for 5 min, the plates were monitored at 580 nm on
a PowerWave XS universal microplate spectrophotometer (Bio-Tek
instruments Inc., Vermont). For library screening, each 96-well plate
contained a positive control using ME and a negative control with no
enzyme. The positive clones were picked from the corresponding
spots on the agar plate.
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Synthesis of NFCD and NCD. A reaction mixture containing
NMN (150 mg, 0.45 mM), Ph P (0.396 g, 1.5 mM), (PyS) (0.33 g,
Activity and Kinetic Assay of ME and Its Variants.
3
2
1
(
5
.5 mM), and L-methylimidazole (0.48 mL, 6 mM) in DMF/DMSO
1:2, 10 mL) was held at room temperature for 15 min. A solution of
-fluorocytidine monophosphate (0.9 mM) in 2 mL DMF was added.
Overexpression and purification of recombinant MEs were performed
28
as previously described. The activity assays were carried out at 25 °C
in 1 cm cuvettes using UV−vis spectrophotometer model V-53
(JASCO, Tokyo, Japan). The activity was measured in 50 mM of
HEPES and pH of 7.2 and in the presence of 3 mM of L-malate and 5
The mixture was stirred at room temperature for 50 min. To the
mixture was added acetone (100 mL). The precipitates were collected
and washed twice with acetone. Purification of the product was
performed by anion exchange column chromatography using 201 × 4
mM of MnCl at varying concentrations of NAD or NFCD. After
2
adding the enzyme solution into the reaction mixture, the absorbance
at 340 nm was continuously monitored for 1 min. One enzyme unit is
defined as the amount of enzyme that catalyzes the formation of 1 μM
of NADH or NFCDH per minute under the assay conditions. A molar
type anion resin (HCO2− form) and eluted with 50 mM HCO H.
2
Fractions containing the product were concentrated and further
purified on a DEAE Sephadex G-25 column using 10 mM NH HCO
4
3
−
1
−1
as the elution buffer. Fractions were pooled, lyophilized, dissolved in
water, and lyophilized to give NFCD as a white solid (145 mg, yield
absorption coefficient of 6220 M cm for NADH, NFCDH, or
NCDH was used in the calculations.
1
+
4
4
5%). H NMR (D O, 400 MHz, NH form): δ 9.17 (s, 1H), 9.02 (d,
For kinetic assay, the Michaelis−Menten constants V and K of
2
max
m
J = 6.1 Hz, 1H), 8.72 (d, J = 7.9 Hz, 1H), 8.05 (m, 1H), 7.70 (d, J =
.4 Hz, 1H), 5.94 (d, J = 5.5 Hz, 1H), 5.55 (d, J = 3.8 Hz, 1H), 4.33
brs, 1H), 4.28 (brs, 1H), 4.19 (brs, 1H), 4.15−4.12 (m, 1H), 4.03−
NAD, NFCD, and NCD of the wild-type and mutant ME were
obtained from initial rate measurements under conditions in which
6
(
3
coenzyme was varied at five different levels from below their Km to
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+
2+
.93 (m, 5H), 3.91−3.89 (m, 1H). C NMR (D O, 100 MHz, NH
above their K . The L-malate (5 mM) and Mn (5 mM) were present
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4
m
form): δ 165.2, 158.0, 157.8, 155.3, 145.9, 142.4, 139.8, 138.5, 136.0,
at a saturated and constant concentration. The experimental data were
fitted with the Michaelis−Menten equation using OriginLab 8.0.
Reactions were incubated in 50 mM of HEPES and pH of 7.5 at 25 °C.
All assays were performed in duplicates.
Constructing DLDH and MDH Expression Plasmid and
Mutagenesis. The gene of DLDH (GenBank No. CAA47255.1)
from L. helveticus was synthesized and inserted between NdeI and XhoI
in the pET24b vector (Novagen) with a C-terminal Leu-Glu as a
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33.7, 128.6, 125.5, 125.2, 99.8, 89.5, 86.6, 82.3, 77.4, 74.0, 70.3, 69.0,
4.8, 64.7. 19F NMR (D O, 376 MHz, NH form): δ −164.9.
+
31
P
2
4
+
NMR (D O, 162 MHz, NH4 form): δ −11.1, −11.2. HRMS:
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+
calculated for C H FN O P (M + H) 658.0963; found 658.0961.
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15 2
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NCD (38 mg, 11%) as a white powder was prepared similarly. H
+
NMR (D O, 400 MHz, NH4 form): δ 9.30 (s, 1H), 9.14 (d, J = 6.1
2
Hz, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.16 (m, 1H), 7.74 (d, J = 7.6 Hz,
1H), 6.04 (d, J = 5.3 Hz, 1H), 5.92 (d, J = 7.4 Hz, 1H), 5.77 (d, J = 4.0
spacer between the protein and the His tag. The gene of MDH
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Hz, 1H), 4.46 (brs, 1H), 4.41 (brs, 1H), 4.33 (brs, 1H), 4.28−4.25 (m,
(GenBank no. CAA68326.1) was isolated form the E. coli genome and
inserted similarly, as was done for DLDH. The mutants DLDH-V152R
and MDH L6R were created by a method as described in the
QuikChange site-directed mutagenesis kit (Invitrogen).
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1
H), 4.17−4.09 (m, 5H), 4.02−4.00 (m, 1H). C NMR (D O, 100
2
+
MHz, NH4 form): δ 165.5, 165.2, 156.4, 146.0, 142.5, 141.7, 139.9,
33.9, 128.6, 99.9, 89.4, 87.0, 82.5, 77.6, 74.1, 70.7, 69.2, 64.9, 64.7. P
NMR (D O, 162 MHz, NH4 form): δ −11.1, −11.3. HRMS:
calculated for C H N O P (M + Na) 662.0877; found 662.0883.
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1
1
+
Activity Assay of DLDH, MDH, and Their Mutants. The
specific activity of DLDH was measured at 25 °C in 1 cm cuvettes
using the UV−vis spectrophotometer. The activity assays were
performed in a total volume of 0.2 mL. The reaction buffer contained
50 mM of HEPES, pH of 7.5, 200 mM of D-lactate, and 1 mM of
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+
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15 2
Construction of Mutant Libraries. Two-site saturation mutant
15
libraries were constructed according to previous methods. To
construct the libraries Q401/L404, L310/Q401, and L310/L404,
degenerated primer (K = G or T; M = C or A; N = A, C, G, or T)
pairs 5′-GTCTCAGGANNKACCGGGNNKTTTACGGAAGAGAT
CATCCG-3′ and 5′-TTCCGTAAAMNNCCCGGTMNNTCCTGA-
GACGCCAATCAG-3′, 5′-ATCGTCTTCNN KGGTGCAGGTT-
CAGC-3′ and 5′-ACAGCCCGGTMNNT CCTGAGACGCCAAT-
CAGAATATC-3′, and 5′-ATCGTCTT CNNKGGTGCAGGTT-
−1
NFCD or NAD. After adding 1 μL of DLDH solution (0.1 mg mL )
into the reaction mixture, the absorbance at 340 nm was continuously
monitored for 1 min, and data were processed as described.
The specific activity of MDH was measured, as was done for
DLDH. The reaction buffer contained 50 mM of Tris−HCl, pH of 8.0,
100 mM of L-malate, and 1 mM of NFCD or NAD.
C A G C - 3 ′
a n d
5 ′ - T C T T C C G T A A A M
Cofactor Recycling. The cofactor recycling experiment with ME-
L310R/Q401C and DLDH-V152R was performed in 50 mM of
HEPES and pH of 7.5, which contained 5 mM of L-malate, 2.5 mM of
NNCCCGGTCTGTCCTGAGAC-3′, respectively, were used. Frag-
ments containing mutagenic modifications were obtained by PCR
using the corresponding primer pairs and the plasmid pET24b-ME as
the template. The resulted DNA fragments were used as megaprimer
to amplify the plasmid pET24b-ME to generate nicked plasmid DNA
containing the corresponding mutations. The PCR mixtures were
treated with DpnI (New England Biolabs, Beijing, China) to digest the
methylated parental plasmid and transformed into electrocompetent E.
coli BL21 (DE3) cells to get the expression libraries.
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MnCl , 0.2 mM of NFCD, 0.002 mg mL of DLDH-V152R, and
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0.008 mg mL of ME-L310R/Q401C. The reaction mixtures were
incubated at 25 °C for 45 min. The concentrations of lactate, pyruvate,
and malate were determined by ICS-2500 ion chromatography system
(Dionex, Sunnyvale, California), equipped with a guard column
IonPac AG11-HC (50 mm ×4 mm), an IonPac AS11-HC analytic
column (250 mm ×4 mm), and an ED50 conductivity detector. All
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dx.doi.org/10.1021/ja2074032 | J. Am. Chem.Soc. 2011, 133, 20857−20862